Analgesic agent for newborn or fetal subjects

ABSTRACT

In a first aspect, the present invention relates to the use of xenon in the preparation of a medicament for providing analgesia in a newborn subject and/or a fetal subject. In a second aspect, the invention relates to a method of providing analgesia in a newborn subject, the method comprising administering to the subject a therapeutically effective amount of xenon. In a third aspect, the invention relates to a method of providing analgesia in a fetal subject, the method comprising administering to the mother of the fetal subject a therapeutically effective amount of xenon for both the mother and fetal subject.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a 371 application of PCT/GB03/03391, filed onAug. 5, 2003, which claims priority to GB application Ser. No.0218145.5, filed on Aug. 5, 2002.

The present invention relates to the field of analgesia. Morespecifically, the invention relates to an analgesic agent suitable foruse in newborn and/or fetal subjects.

BACKGROUND

The human fetus and newborn are known to experience pain sensation[Anand K J S et al, New Engl J Med 1987; 317:1321-1329; Fitzgerald M, BrMed Bull 1991;47:667-75]. However, of greater concern is that untreatedpain in the newborn may adversely affect development of the centralnervous system resulting in long-term physiological and psychologicalconsequences [Taddio A et al, Lancet 1997; 349:599-603; Graham YP et al,Dev Psychopath 1999; 11:545-565; Anand K J S et al, Biol Neonate 2000;77:69-82; Ruda M A et al, Science 2000; 289:628-630]. As a consequence,appropriate analgesic therapy is even more important in the anaestheticmanagement of the very young than in adults.

Nitrous oxide (N₂O) has been used for clinical anaesthesia in the youngand the old for more than 150 years and remains the most commonly usedanaesthetic gas. N₂O usage in the paediatric surgical patient is basedupon the assumption that its anaesthetic and analgesic efficacy matchesthat seen in adults [Eger E L, Nitrous Oxide/N₂O; Elsevier, New York,1985]. However, the expectation that efficacious analgesic drugs inadults will exert the same beneficial effects in neonates has beenchallenged by our recent report that nitrous oxide (N₂O) is ineffectivein neonatal rats because the immature pain pathways cannot activate thedescending inhibitory pathway in response to nociceptive stimuli[Fitzgerald M et al, Brain Res 1986;389:261-70; van Praag H, Frenk H,Dev Brain Res 1991;64:71-76]. Experiments have shown that N₂O lacksantinociceptive effects against thermal [Fujinaga M et al, Anesth Analg2000; 91:6-10] and inflammatory [Ohashi Y et al, Pain 2002; 100:7-18]stimulation in rats under 3 weeks of age. If extrapolatable to humans,this would mean that N₂O is ineffective as an analgesic agent insubjects up to and including the toddler stage. A similar rationale wasthought to apply to the use of xenon as an analgesic agent.

The present invention seeks to provide an analgesic agent capable ofproviding effective pain relief in newborn and/or fetal subjects whichalleviates one or more of the above-mentioned problems.

STATEMENT OF INVENTION

In a first aspect, the present invention relates to the use of xenon inthe preparation of a medicament for providing analgesia in a newbornsubject and/or a fetal subject.

In a second aspect, the invention relates to a method of providinganalgesia in a newborn subject, the method comprising administering tothe subject a therapeutically effective amount of xenon.

In a third aspect, the invention relates to a method of providinganalgesia in a fetal subject, the method comprising administering to themother of the fetal subject a therapeutically effective amount of xenon.

DETAILED DESCRIPTION

As mentioned above, in a broad aspect, the present invention relates tothe use of xenon as an analgesic agent in newborn and/or fetal subjects.

More specifically, the invention relates to the use of xenon in thepreparation of a medicament for providing analgesia in a newbornsubject.

Surprisingly, it has been found that xenon is capable of providingeffective analgesia in the newborn, despite prior art indications to thecontrary. Indeed, it is to be noted that the prior art has neitherdisclosed nor suggested the use of xenon as an analgesic agent inneonatal subjects.

In a preferred embodiment, the newborn subject is a mammal in the firstfour weeks after birth. More preferably, the newborn subject is a mammalin the first two weeks, more preferably still, the first week afterbirth.

Even more preferably, the newborn subject is a human.

Xenon is a chemically inert gas whose anaesthetic properties have beenknown for over 50 years [Lawrence J H et al, J. Physiol. 1946;105:197-204]. Since its first use in surgery [Cullen S C et al, Science1951; 113:580-582], a number of research groups have shown it has anexcellent pharmacological profile, including the absence of metabolicby-products, profound analgesia, rapid onset and recovery, and minimaleffects on the cardiovascular system [Lachmann B et al, Lancet 1990;335:1413-1415; Kennedy R R et al, Anaesth. Intens. Care 1992; 20:66-70;Luttropp H H et al, Acta Anaesthesiol. Scand. 1994; 38:121-125; Goto Tet al, Anesthesiology 1997; 86:1273-1278; Marx T et al, Br. J. Anaesth.1997; 78:326-327].

It has recently been discovered that xenon (which rapidly equilibrateswith the brain) is an NMDA antagonist [Franks N P et al, Nature 1998;396:324]. Mechanistic studies on cultured hippocampal neurons have shownthat 80% xenon, which will maintain surgical anaesthesia, reducesNMDA-activated currents by up to 60%. This powerful inhibition of theNMDA receptor explains some of the important features of thepharmacological profile and is likely to be instrumental in theanaesthetic and analgesic effects of this inert gas.

The use of xenon in a pharmaceutical application is described in WO00/76545, while the use of xenon as a neuroprotectant is described in WO01/08692, the contents of which are incorporated herein by reference.Neither patent application discloses the possibility of xenon being aneffective analgesic for newborn or fetal subjects.

The advantage of using an inert, volatile gas such as xenon as ananalgesic agent is that the molecule can be rapidly eliminated viarespiration. Xenon is currently thought to be a potential replacementfor N₂O [Rossaint R et al, Anesthesiology 2003;98:6-13]. In humans,xenon has a minimum alveolar concentration (MAC) of 71% atm [Lynch C etal, Anesthesiology 2000;92:865-70] which is even lower in elderly femalepatients (51% atn) [Goto T et al, Anesthesiology 2002;97:1129-32] andthus is more potent than N₂O (MAC=104%) [Hornbein T F et al, Anesth.Analg. 1982;61:553-6]. Xenon has faster induction and emergence [Goto Tet al, Anesthesiology 1997;86:1273-8; Rossaint R et al, Anesthesiology2003;98:6-13] due to its very low blood gas partition coefficient(0.115) [Goto T et al, Br J Anaesth 1998;80: 255-6], is devoid ofteratogenic effects [Lane G A et al, Science 1980;210:899-901; Burov N Eet al, Anesteziol Reanimatol 1999;6:56-60], is less harmful to theenvironment [Goto T, Can J Anaesth 2002:49: 335-8], and exhibits a lowerrisk of diffusion hypoxia [Calzia E et al, Anesthesiology1999;90:829-3].

Studies by the applicant investigated the efficacy of xenon againstformalin-induced nociception as reflected by behaviour and c-Fosexpression (a marker of neuronal activation) in cohorts of rats atvarious ages. Further details of these experiments are outlined in theaccompanying Examples.

In brief four cohorts of Fischer rats aged, 7, 19, 28 and greater than77 days (adult), were exposed to either air or 70% xenon. Formalinplantar testing was used to mimic surgical stimulation, and this wasassessed using immunohistochemical (c-Fos staining) and behavioralmethods. Formalin administration produced a typical nociceptive responseobserved both behaviorally and immunohistochemically in each age groupduring exposure to air. However, these responses were significantlyattenuated by xenon; in other words, xenon was shown to exert anantinociceptive response against formalin injection in Fischer rats ateach of four developmental stages, i.e. at days 7, 19 and 28 day as wellas in adults. These data are qualitatively different from those recentlyreported with N₂O [Ohashi Y et al, Pain 2002;100:7-18] in which noantinociceptive effect (neither behaviorally nor immunohstochemically)was noted in animals younger than 23 days old.

The present invention further relates to the use of xenon in thepreparation of a medicament for providing analgesia in a fetal subject.In this embodiment of the invention, the xenon is preferablyadministered to the mother prior to or during labour.

During birthing, the fetus is subjected to mechanical stress whichresults in the activation of pain pathways. The present inventiondemonstrates that the impact of the activation of pain processingpathways in fetal subjects can be mitigated by the administration ofxenon.

It is notable that to date, there has been no teaching or suggestion inthe prior art to indicate that xenon could be used to provide analgesiain fetal subjects.

In one preferred embodiment, the xenon is used in combination with oneor more other pharmaceutically active agents. The agent may be anysuitable pharmaceutically active agent including anaesthetic or sedativeagents which promote GABAergic activity. Examples of such GABAergicagents include isoflurane, propofol and benzodiazapines.

The xenon may also be used in combination with one or more otheranalgesic agents. Suitable analgesic agents may include alpha-2adrenergic agonists, opiates or non-steroidal antiinflammatory drugs.Examples of suitable alpha-2 adrenergic agonists include clonidine,detomidine, medetomidine, brimonidine, tizanidine, mivazerol, guanabenz,guanfacine or dexmedetomidine.

The medicament of the present invention may also comprise other activeingredients such as L-type calcium channel blockers, N-type calciumchannel blockers, substance P antagonists, sodium channel blockers,purinergic receptor blockers, or combinations thereof.

In one highly preferred embodiment of the invention, the xenon isadministered by inhalation. More preferably, the xenon is administeredby inhalation of a 20-70% v/v xenon/air mixture.

In another preferred embodiment, the medicament is in liquid form. Forparenteral administration, the medicament may be used in the form of asterile aqueous solution which may contain other substances, for exampleenough salts or monosaccharides to make the solution isotonic withblood.

In a more preferred embodiment, the xenon is used in combination with apharmaceutically acceptable carrier, diluent or excipient.

Acceptable carriers or diluents for therapeutic use are well known inthe pharmaceutical art, and are described, for example, in Remington'sPharmaceutical Sciences, Mack Publishing Co. [A. R. Gennaro edit. 1985].

The choice of pharmaceutical carrier, excipient or diluent can beselected with regard to the intended route of administration andstandard pharmaceutical practice. Examples of suitable carriers includelactose, starch, glucose, methyl cellulose, magnesium stearate,mannitol, sorbitol and the like. Examples of suitable diluents includeethanol, glycerol and water.

The medicament may comprise as, or in addition to, the carrier,excipient or diluent any suitable binder(s), lubricant(s), suspendingagent(s), coating agent(s), solubilising agent(s). Examples of suchsuitable excipients for the various different forms of pharmaceuticalcompositions described herein may be found in the “Handbook ofPharmaceutical Excipients, 2^(nd) Edition, (1994), Edited by A Wade andP J Weller.

Preservatives, stabilizers, dyes and even flavoring agents may beprovided in the pharmaceutical composition. Examples of preservativesinclude sodium benzoate, sorbic acid and esters of p-hydroxybenzoicacid. Antioxidants and suspending agents may be also used.

Up to now, a significant problem which has impeded the use of xenon as anew anaesthetic is its high cost and the need to use complex apparatusto minimise the volume used (low-flow systems), along with the need toscavenge the gas for reuse. A further problem is that the potency ofxenon is relatively low. As a consequence, it had been suggested thatvolatile general anaesthetics may be solubilised in a lipid emulsion andadministered intravenously [Eger R P et al, Can. J. Anaesth. 1995;42:173-176]. It is known in the art that local anaesthesia can beinduced by intradermally injecting microdroplets of a generalanaesthetic in a liquid form [Haynes D H, U.S. Pat. Nos. 4,725,442 and44,622,219]. Typically these microdroplets are coated with aunimolecular phospholipid layer and remain stable inphysiologically-compatible solutions. A similar approach is described ina recent patent application which proposes that xenon might beadministered in this fashion [Georgieff M, European Patent ApplicationNo. 864329-A1].

Thus, in an even more preferred embodiment, the medicament is in theform of a lipid emulsion. By way of example, an intravenous formulationtypically contains a lipid emulsion (such as the commercially availableIntralipid®10, Intralipid®20, Intrafat®, Lipofundin®S or Liposyn®emulsions, or one specially formulated to maximise solubility) tosufficiently increase the solubility of the gas or volatile anaestheticto achieve the desired clinical effect. Further information on lipidemulsions of this sort may be found in G. Kleinberger and H. Pamperl,Infusionstherapie, 108-117 (1983) 3.

The lipid phase of the present invention which dissolves or dispersesthe gas is typically formed from saturated and unsaturated long andmedium chain fatty acid esters containing 8 to 30 carbon atoms. Theselipids form liposomes in aqueous solution. Examples include fish oil,and plant oils such as soya bean oil thistle oil or cottonseed oil. Thelipid emulsions of the invention are typically oil-in-water emulsionswherein the proportion of fat in the emulsion is conventionally 5 to 30%by weight, and preferably 10 to 20% by weight. Oil-in-water emulsions ofthis sort are often prepared in the presence of an emulsifying agentsuch as a soya phosphatide.

The lipids which form the liposomes of the present invention may benatural or synthetic and include cholesterol, glycolipids,sphingomyelin, glucolipids, glycosphingolipids, phosphatidylcholine,phosphatidylethanolamine, phosphatidylserine, phosphatidyglycerol,phosphatidylinositol.

The lipid emulsions of the present invention may also compriseadditional components. These may include antioxidants, additives whichmake the osmolarity of the aqueous phase surrounding the lipid phaseisotonic with the blood, or polymers which modify the surface of theliposomes.

It has been established that appreciable amounts of xenon maybe added toa lipid emulsion. Even by the simplest means, at 20° C. and normalpressure, xenon can be dissolved or dispersed in concentrations of 0.2to 10 ml or more per ml of emulsion. The concentration of dissolved gasis dependent on a number of factors, including temperature, pressure andthe concentration of lipid.

The lipid emulsions of the present invention may be loaded with agaseous or volatile anaesthetic. In general, a device is filled with theemulsion and anaesthetics as gases or vapours passed through sinteredglass bubblers immersed in the emulsion. The emulsion is allowed toequilibrate with the anaesthetic gas or vapour at a chosen partialpressure. When stored in gas tight containers, these lipid emulsionsshow sufficient stability for the anaesthetic not to be released as agas over conventional storage periods.

The lipid emulsions of the present invention may be loaded so that thexenon is at the saturation level. Alternatively, the xenon may bepresent in lower concentrations, provided, for example, that theadministration of the emulsion produces the desired pharmaceuticalactivity.

In one preferred embodiment, the medicament is in a form suitable fordelivery intravenously (either by bolus administration or infusion),neuraxially (either subdural or subarachnoid) or transdermally.

The medicament of the present invention may also be administered in theform of an ointment or cream (lipid emulsion or liposomes) appliedtransdermally. For example, the medicament of the present invention maybe incorporated into a cream consisting of an aqueous emulsion ofpolyethylene glycols or liquid paraffin. Alternatively, the medicamentof the present invention may be incorporated, at a concentration ofbetween 1 and 10% by weight, into an ointment consisting of a white waxor white soft paraffin base together with such stabilisers andpreservatives as may be required. These ointments or creams are suitablefor the local alleviation of pain and may be applied directly to damagedtissue, often with the aid of an optionally air-tight wound closure.

The concentrations employed in the medicament formulation may be theminimum concentration required to achieve the desired clinical effect.It is usual for a physician to determine the actual dosage that will bemost suitable for an individual patient, and this dose will vary withthe age, weight and response of the particular patient. There can, ofcourse, be individual instances where higher or lower dosage ranges aremerited, and such are within the scope of this invention.

The medicament of the present invention may be for human administrationor animal administration.

Thus, the medicament of the present invention may also be used as ananimal medicament. In this regard, the invention further relates to theuse of xenon in the preparation of a veterinary medicament for providinganalgesia in newborn animals. Preferably, the medicament of the presentinvention further comprises a veterinarily acceptable diluent, excipientor carrier.

For veterinary use, the medicament of the present invention, or aveterinarily acceptable formulation thereof, is typically administeredin accordance with normal veterinary practice and the veterinary surgeonwill determine the dosing regimen and route of administration which willbe most appropriate for a particular animal.

A further aspect of the invention relates to a method of providinganalgesia in a newborn subject, the method comprising administering tothe subject a therapeutically effective amount of xenon.

Yet another aspect of the invention relates to a method of providinganalgesia in a fetal subject, the method comprising administering to themother of the fetal subject a therapeutically effective amount of xenon.Preferably, the xenon is administered in an amount which istherapeutically effective for both the mother and the fetal subject.

In a preferred embodiment, the xenon is administered to the mother priorto or during labour. Preferably, the xenon alleviates the painassociated with the mechanical stress experienced by the fetus duringlabour.

Advantageously, administering xenon to the fetus via the mother has theconcomitant benefit of alleviating labour pain experienced by the motherduring delivery. Thus, the administration of xenon to the mother priorto or during labour has the dual effect of providing pain relief to boththe fetus and the mother.

The present invention is further described by way of the followingnon-limiting examples and with reference to the following Figures,wherein:

FIG. 1 shows cross sections of the spinal cord at the lumbar levelstained for c-Fos in 7 day-old Fischer rats after receiving formalin.FIG. 1A shows a section treated with air/formalin, whereas FIG. 1B showsa section treated with xenon/formalin.

FIG. 2 shows cross sections of the spinal cord at the lumbar levelstained for c-Fos in 7 day-old Fischer rats after receiving formalin.FIG. 2A shows a section treated with air/formalin, whereas FIG. 2B showsa section treated with N₂O/formalin.

FIG. 3 shows a schematic diagram of the experimental apparatus and gasdelivery closed circle system. This system consisted of an anaestheticchamber, a rubber bag, an air pump and a xenon monitor (Model 439xe, Airproduct, UK).

FIG. 4 shows nociceptive scoring curves from the four aged-groups in 7,19, 28 olds Fischer pups and adults with two sub-treatments(Air+Formalin and Xe+Formalin). The ordinate reflects nociceptiveintensity (lower values indicates less nociceptive behavior). Theabcissa indicates the time period after formalin injection (min). Theclassical biphasic behavioural response to formalin can be seen in thegroup receiving air.

FIG. 5 shows a representative section from the spinal cords at thelumbar level of the spinal cord showing c-Fos response to formalininjection in neonatal rat pups aged 7, 19, 28 days and in an adultreceiving either air (left column) or xenon (right column).

FIG. 6 shows the number (mean±SEM, n=4) of c-Fos positive cells at thelumbar level in response to formalin injection from the four age-groupsanimals receiving either air (black bar) or 70% xenon /20% O₂/10% N₂(Xe) (dot bar) or in response to saline injection from the fourage-groups receiving air (white bar). *P<0.01, **P<0.001 relative toAir+formaline group at the corresponding region. +P<0.01, ++P<0.001relative to Xenon+formalin. The figures in the left column representc-Fos expression ipsilaterally associated with injection and those inthe right column represent c-Fos expression contralaterally associatedwith injection. From 19-day-old to adult, laminae I-II (superficialarea), laminae II-IV (nucleus proprius area), laminae V-VI (neck area)and laminae VII-X (ventral area) in the spinal cord section is presentedby A/B, C, D and E as equivalent to the five regions in 7-day-old pups.

FIG. 7 shows the total number (mean±SEM, n=3) of c-Fos positive cellsper section at the lumbar level of the spinal cord from the fourage-groups animals receiving either air (block bar) or 70% xenon /20%O₂/10% N₂ (Xe) (dot bar). No differences were found between thecorresponding age group.

EXAMPLES Example 1

The analgesic efficacy of xenon was investigated in a neonatal rat pup.A 7 day neonatal rat pup is known to be developmentally equivalent to ahuman full term fetus with respect to pain processing pathways.

A 7 day old rat was injected with formalin into the hindpaw duringexposure to either air or xenon (70% v/v). 90 minutes later the animalwas killed and the spinal cord removed; evidence of activation ofpain-processing pathways by formalin was sought by counting the numberof cFos positive neurones in the dorsal horn of the spinal cord.

In FIG. 1, xenon almost completely attenuated formalin-induced c-Fospositive neurones (air). By comparison a normally analgesic dose ofnitrous oxide in the adult rat did not change formalin-induced c-Fospositive neurones (FIG. 2).

From the results, it can be concluded that xenon interrupts painprocessing so that pain signals will not travel to the brain and hencepain, as well as the long-term consequences of untreated pain, ismitigated in the neonatal population.

Example 2

Materials and Methods

General Procedures and Animals

The study protocol was approved by the Home Office (UK), and all effortswere made to minimize animal suffering and the number of animals used.Fischer rats were used for the entire study (B&K Universal, GrimstonAldbrough Hull, UK). The rats were provided ad libitum food and water,and artificial lighting between 6 a.m. and 6 p.m. The age of each animalwas determined from the body weight, based on a previously establishedgrowth curve (Hashimoto et al., 2002). (The date of birth was defined as0 day-old.) Experiments were performed on rat pups of 7, 19, 28 day-oldand on adult rats (11-12 week-old).

Within each age-group, there were three cohorts (n=3 - 4)“Air+formalin”, “Xenon+formalin” and “Air+saline” cohorts. In theAir+formalin group, animals exposed to air were injected with 5%formalin into the plantar surface of their left hind paw subcutaneously.In the Xenon+formalin group, animals exposed to 70% Xe/20% O₂/10% N₂were injected with 5% formalin as described above. In the Air+salinegroup, animals exposed to air were injected with saline as above. Thevolumes of formalin or saline injected were adjusted for each age groupas previously reported [Ohashi Y et al, Pain 2002;100:7-18] and were asfollows: 10 μl for 7 days old; 15 μl for 19 days old; 20 μl for 28 daysold; 50 μl for adults.

Gas Exposure

A recirculating system for exposure to the gas was constructed tominimise xenon consumption (FIG. 3). The circuit was flushed with gas(either air or xenon/oxygen/nitrogen) at a flow rate of 4 l/min andafter the desired gas concentrations were achieved, the flow rate wasreduced to 40 ml/min for the remainder of the experimental period. Thecarbon dioxide level and the humidity were kept less than 0.6% and 50%respectively with soda lime and silica gel. Formalin or saline wasadministered 15 min after gas exposure; thereafter, animals were exposedto the gas mixture for a further 90 min.

Nociceptive Intensity Scoring

Immediately after injection of formalin, behavior was recorded for 60min with a video camera (MegaPixel, Digital Handycam, Sony) positionedapproximately 50 cm beneath the floor of the chamber to allow anunobstructed view of the paws (visible via a television monitor) and tofacilitate recording of animal behavior. The chamber and holding areafor pups waiting to be tested were maintained at room temperaturethroughout the experiment.

Nociceptive behavior was assessed in the 7-d old pups for the presence(“1”) or absence (“0”) of flexion, shaking, and whole body jerking perepoch of time [Teng C J et al, Pain 1998;76:337-47] and calculated as[Nociceptive score=T/300, where T is the duration (sec.) of nociceptivebehavior exhibited during consecutive 300 sec post-injection epochs.]

Older rat pups were scored across four categories of pain behavior afterxenon administration: no pain (the injected paw was in continuouscontact with floor=“0”), favoring (the injected paw rested lightly onthe floor=“1”), lifting (the injected paw was elevated all the time=“2”)and licking (licking, biting or shaking of the injected paw=“3”) [Teng CJ et al, Pain 1998;76:337-47] and calculated as [Nociceptive score=(T1+[T2×2]+[T3×3])/300, where T1, T2 and T3 are the durations (sec)spent in categories 1, 2 or 3 per 300 sec epoch].

Immunohistochemical Staining and Quantitative Counting of c-Fos

Ninety min after the formalin injection, animals were deeplyanesthetised with pentobarbital (100 mg/kg, i.p.) and perfused with 4%paraformaldehyde. The whole spinal cord was removed. The lumbarenlargement was sectioned transversely at 30 μm and then was stained forc-Fos as previously described [Ma D et al, Br J Anaesth 2002;89:73946].Briefly, sections were incubated for 30 min in 0.3% H₂O₂ in methanol andthereafter washed three times in 0.1M phosphate buffered saline (PBS).Following this, the sections were incubated for 1 hour in a “blockingsolution” consisting of 3% donkey serum and 0.3% Triton X in PBS (PBT)and subsequently incubated overnight at 4° C. in 1:5,000 goat anti-c-Fosantibody (sc-52-G, Santa Cruz Biotechnology, Santa Cruz, Calif.) in PBTwith 1% donkey serum. The sections were then rinsed 3 times with PBT andincubated with 1:200 donkey anti-goat IgG (Vector laboratories,Burlingame, Calif.) in PBT with 1% Donkey serum for 1 hour. The sectionswere washed again with PBT and incubated with avidin-biotin-peroxidasecomplex (Vector Laboratories) in PBT for 1 hour. The sections wererinsed 3 times with PBS and stained with 3,3′-diaminobenzidine (DAB)with nickel ammonium sulphate in which hydrogen peroxide was added (DABkit, Vector Laboratories). After the staining was completed, thesections were rinsed in PBS followed by distilled water and mounted,dehydrated with 100% ethanol, cleaned with 100% xylene and covered withcover slips.

Photomicrographs of three sections per each animal were scored for c-Fospositive neurons by an observer who was blinded to the experimentaltreatment. For the purpose of localizing the c-Fos positive cells tofunctional regions of the spinal cord, each section was divided into A/B(laminae I-II or the superficial area), C (laminae II-IV or nucleusproprius area), D (laminae V-VI or the neck area and E (laminae VII-Xorthe ventral area) [Yi D K et al, Pain 1995;60:257-265].

Data Analysis

The nociceptive intensity scoring against time in each animal wasplotted and the area under curve (over a 60 min time period) (AUC) fromeach animal was calculated. The mean of c-Fos positive neurons for threerepresentative sections in each region as described above was theaggregate score for each animal. The results of nociceptive intensity orc-Fos positive neurons are reported as means±SEMs. The statisticalanalysis was performed by one-way analysis of variance, followed byNewman-Keuls test. A p value <0.05 was regarded as statisticallysignificant.

Results

Behavioral Nociceptive Response

The time course of the nociceptive response of each cohort in each agecategory is presented in FIG. 4. Following injection with saline, theanimals exposed to air exhibited a non-specific nociceptive behavior(score 1) involving the injected paw for a period of approximately twominutes duration. A biphasic nociceptive response is induced by formalininjection in each of the age groups administered air. The AUC data arepresented in Table 1.

During the pre-injection period, 7 day-old rats exposed to air wereawake and active. Following injection with formalin, the animalsexhibited intense nociceptive behaviour (violent kicking, flexion andshaking of the injected paw) for up to 50 minutes but the exhibition ofpainful behavior appeared to be less than that seen with adult animals.Those rats exposed to xenon exhibited only mild nociceptive behaviourfor the first two minutes after formalin injection followed by nofurther movement for the rest of the 60-min observation period. The AUCfor the group exposed to xenon was significantly different from thegroup exposed to air (P<0.001; Table 1).

Following injection with formalin, the 19 day-old animals exposed to airexhibited intense nociceptive behaviour, which was biphasic, persistingfor the majority of the observation period before gradually decreased(FIG. 4: 19 day old). There was a significant decrease in nociceptivebehaviour exhibited by the animals that were exposed to xenon (P<0.01)(Table 1). In the 28 day-old cohort, animals exposed to air exhibitedbiphasic nociceptive behaviour; in the presence of xenon the nociceptivebehaviour was significantly less intense (P<0.001) (Table 1). Thenociceptive behaviour exhibited by adult rats in response to formalinwas less intense with xenon than air exposure (P<0.001) (Table 1).

Immunohistochemical Nociceptive Response

Nociceptive Stimuli Induced c-Fos Expression

Formalin-induced c-Fos expression at the lumbar level of the spinal cordipsilateral to the site of injection increased in all age groups.Exposure to xenon significantly suppressed c-Fos expression. In the 7day-old pups, xenon exposure reduced c-Fos expression in response toformalin by 48% in laminae A/B (P<0.001), by 50% in lamina C (P<0.001),by 50% in lamina D (P<0.001) and by 28% in lamina E (P<0.01). In the 19day-old rats xenon suppressed mean c-Fos expression in response to xenonby 55% in laminae I-II (P<0.001), by 57% in lamina III-IV (P<0.001) andby 62% in lamina V-VI (P<0.001). In the 28 day-old rats, xenon depressedc-Fos expression in response to formalin by 34% in laminae I-II(P<0.001), by 27% in lamina III-IV (P<0.001) and by 28% in lamina V-VI(P <0.001). In adult rats xenon inhibited c-Fos expression by 41% inlaminae I-II (P<0.001), by 45% in lamina III-IV (P<0.001) and by 34% inlamina V-VI (P<0.001). Saline injection also caused c-Fos expressionipsilateral to the injection; however, this was much less intense thanthat induced by formalin injection (FIGS. 5 and 6).

Control Study

In order to test whether xenon itself can cause c-Fos expression (as isthe case with nitrous oxide) [Hashimoto T et al, Anesthesiology2001;95:463-9], naïve animals were exposed to either air or the xenonmixture gas (70% Xe/20% O₂/10% N₂) for 90 min (FIG. 7). The number ofc-Fos positive cells did not differ between these groups in any regionof the spinal cord.

Discussion

The present study demonstrates that xenon exerts an antinociceptiveresponse against formalin injection in Fischer rats at each of fourdevelopmental stages, i.e. at days 7, 19 and 28 day as well as inadults. These data are qualitatively different from those recentlyreported with N₂O [Ohashi Y et al, Pain 2002;100:7-18] in which noantinociceptive effect (neither behaviorally nor immunohstochemically)was noted in animals younger than 23 days old.

In various in vitro preparations xenon and N₂O are known to exertsimilar effects at nicotinic acetylcholine [Yamakura T et al,Anesthesiology 2000;93:1095-101], serotonin 3A [Suzuki T et al,Anesthesiology 2002;96:699-704], GABA [Yamakura T et al, Anesthesiology2000;93:1095-101; Mennerick S et al, J. Neurosci 1998;18:9716-26], andglycine receptors [Daniels S et al, Toxicol. Lett. 1998;100-101:71-6]and both xenon [Franks N P et al, Nature 1998;396:324; de Sousa S L etal, Anesthesiology 2000;92:1055-66] and N₂O [Jevtovic-Todorovic V et al,Nat Medicine 1998;4:460-63] are inhibitors of the NMDA receptor.Previous studies revealed that xenon and N₂O suppressed wide dynamicrange (WDR) neurons within the intact spinal cord [Utsumi J et al,Anesth Analg. 1997;84:1372-6]. However, xenon exhibited a greaterinhibitory effect on these neurons in spinal cord-transected preparationthan was seen after N₂O exposure [Miyazagi Y et al, Anesth Analg1999;88:893-7]. These studies suggest that xenon directly inhibits WDRneurons at the spinal level to produce antinociception while N₂O-inducedantinociception requires involvement of higher supra-spinal centres[Fujinaga M et al, Mol Neurobiol. 2002;25:167-89]. N₂O modulatesnociception primarily by activation of noradrenergic descendinginhibitory neurons from supra-spinal centres with little direct action,at the level of the spinal cord [Fujinaga M et al, Mol Neurobiol.2002;25:167-89]. Given the evidence for an absolute requirement forfunctional connectivity between the spinal and supra-spinal regions forN₂O-induced antinociception, the applicants predicted and subsequentlyconfirmed that N₂O does not exhibit antinociception [Ohashi Y et al,Pain 2002;100:7-18] before development of such connectivity, i.e., below23 days of age [Fitzgerald M et al, Brain Res 1986;389:261-70]. Thus, incontrast to xenon, N₂O is not an effective antinociceptive agent in theneonatal age group.

The NMDA subtype of the glutamate receptor has been implicated in thenociceptive response to most inflammatory models of pain including thatinduced by formalin [Malmberg A B et al, Pain 2003;101:109-16]. As bothxenon and N₂O are NMDA antagonists, the reason for the qualitativedifferences in antinociception that exists between these two compoundsis unclear. One possible explanation is that different NMDA receptorsubunit combinations have different sensitivities to xenon or N₂O. Humanendogenous NMDA receptors are composed of a combination of NR1 and NR2or NR3 subunits [Dingledine R et al, Pharmacol Rev 1999;51:7-61]. Ofrelevance, the NR2B subunit is postulated to mediate nociceptivetransmission in the dorsal horn of the spinal cord [Boyce S et al,Neuropharmacology 1999;38:611-23] and forebrain [Wei F et al, NatNeurosci 2001;4:164-9]. Of note NR2B antagonists have been linked tosedation [Chizh B A et al, Neuropharmacology 2001;40:212-20] and xenonis a more potent sedative-hypnotic agent than N₂O [Lynch C et al,Anesthesiology 2000;92:865-70] with a MAC-awake of 33% [Goto T et al,Anesthesiology 2000;93:1188-93].

Based on these findings, xenon is expected to be an effectiveantinociceptive agent from a very early age in humans. Xenon's safetyprofile has yet to be examined in the very young, although it is aremarkably safe anesthetic in adults [Rossaint R et al, Anesthesiology2003;98:6-13]. A major cause for concern in the clinical use of NMDAantagonists is their inherent neurotoxicity [Olney J W et al, Science1989;244:360-2; Olney J W et al, Science 1991;254:1515-8], but this doesnot appear to exist with administration of xenon [Ma D et al, Br JAnaesth 2002;89:739-46], a similarity shared with NR2B selective NMDAantagonists [Gill R et al, J Pharmacol Exp Ther. 2002;302:940-8].

By way of summary, formalin administration produces a typicalnociceptive response observed both behaviorally andimmunohistochemically ipsilateral to the site of injection in each agegroup tested. Xenon suppresses both the behavioural and theimmunohistochemical nociceptive responses even in very young animals.Unlike N₂O, the antinociceptive effect of xenon does not appear torequire functional connectivity between the supra-spinal and spinal painprocessing pathways.

Various modifications and variations of the described methods of theinvention will be apparent to those skilled in the art without departingfrom the scope and spirit of the invention. Although the invention hasbeen described in connection with specific preferred embodiments,various modifications of the described modes for carrying out theinvention which are obvious to those skilled in chemistry or relatedfields are intended to be within the scope of the following claims.

TABLE 1 The area under curve (AUC) (mean ± SEM, n = 3-4) was calculatedfrom nociceptive intensity scoring curves (FIG. 4). Air + formalin 70%Xe + formalin Air + saline  7-day-old   11(0.4) 0.17(0.04)**0.15(0.04)** 19-day-old 88(8)  5(0.9)* 4.6(1.2)*  28-day-old 81(7)1.0(0.3)**   5(0.5)** Adult  120(0.7) 0.8(0.1)** 0.4(0.1)** *P < 0.01,**P < 0.001 relative to Air + formalin group at the correspondingaged-group

1. A method of providing analgesia in a human newborn subject, themethod comprising administering a therapeutically effective amount of ananalgesic to a human newborn subject experiencing pain or stresssufficient to necessitate administration of the analgesic, wherein theanalgesic is xenon.
 2. The method of claim 1 wherein the newborn subjectis a mammal in the first four weeks after birth.
 3. The method of claim1 wherein the xenon is administered in combination with a sedative, ananaesthetic agent or a further analgesic agent.
 4. The method of claim 1wherein the medicament is in gaseous form.
 5. The method of claim 1wherein the medicament is in liquid form.
 6. A method accordingly toclaim 1 wherein the xenon is administered in combination with apharmaceutically acceptable carrier, diluent or excipient.
 7. A methodaccording to claim 1 wherein the xenon is administered in the form of a20 to 70% v/v xenon/air mixture.
 8. A method according to claim 1wherein the xenon is administered in the form of a lipid emulsion.
 9. Amethod according to claim 1 wherein the xenon is administeredintraveneously, neuraxially or transversally.
 10. A method of providinganalgesia in a human fetal subject, the method comprising administeringa therapeutically effective amount of an analgesic to a mother of ahuman fetal subject, the fetal subject experiencing pain or stresssufficient to necessitate administration of the analgesic, wherein theanalgesic is xenon.
 11. A method accordingly to claim 1 wherein thexenon is administered in combination with a pharmaceutically acceptablecarrier, diluent or excipient.
 12. A method according to claim 10wherein the xenon is administered in the form of a 20 to 70% o v/vxenon/air mixture.
 13. A method according to claim 10 wherein the xenonis administered in the form of a lipid emulsion.
 14. A method accordingto claim 10 wherein the xenon is administered intraveneously,neuraxially or transdermally.
 15. The method according to claim 10,wherein the xenon is administered to the mother prior to labor.
 16. Themethod according to claim 10, wherein the xenon is administered to themother during labor.
 17. The method of claim 3, wherein the furtheranalgesic agent is an alpha-2-adrenergic agonist, an opiate, or anon-steroidal anti-inflammatory drug.
 18. The method of claim 3, whereinthe sedative or anaesthetic agent promotes GABAergic activity.